Hematopoietic stern cells (HSC), unlike e.g. embryonic stem cells, have a finite potential to divide. Limitations in the replication potential of HSC appear to be important in hematological disorders such as aplastic anemia and chronic myeloid leukemia. Such limitations could furthermore hamper the development of novel therapeutic strategies, including ex vivo stem cell expansion and gene therapy. Based on these considerations, studies that may help define and extend the replicative potential of HSC are important and a general interest. Previous studies with purified human """"""""candidate"""""""" HSC funded by this grant have shown that the functional properties of HSC change dramatically during ontogeny and that the loss in HSC proliferative potential with age correlates with measurable shortening of telomeres. Here we propose to further examine the role of telomerase and telomeres in hematopoiesis. Specifically, we want to test the hypothesis that the replication history of HSC can be traced by studies of their telomere length. In order to test this hypothesis, we will examine the telomere length in subsets of purified HSC and their cultured progeny relative to defined populations of more mature cells using refined flow cytometry techniques developed in our laboratory. These techniques will also be used to further study the age related decline in telomere length in nucleated blood cells from normal individuals, patients with various hematological disorders and pedigrees of genotyped normal baboons. We will furthermore attempt to manipulate the telomere length in HSC using gene transfer and protein transduction strategies and study the functional properties of cells with extended telomeres in vitro and in vivo.
The specific aims are:1) To study the telomere length in purified HSC and nucleated blood cells from normal individuals and patients with various hematological disorders before and after therapy.2) To study the telomere length in nucleated blood cells from baboons in relation to their age and genotype.3) To study the effect of artificial telomere elongation and telomerase inhibition on the proliferation, differentiation and replicative potential of purified """"""""candidate"""""""" HSC in vitro and in vivo.Taken together, these studies will provide crucial baseline information on the role of telomeres in the biology of HSC. Such information is relevant for a basic understanding of hematopoiesis as well as applications of stem cells in and outside hematology.
